MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis

Background: MicroRNAs have recently been recognized to be engaged in the development of bone diseases. Objective: This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. Method: The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. Results: ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased β-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. Conclusion: MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

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MicroRNA-132, Delivered by Mesenchymal Stem Cell-Derived Exosomes, Promote Angiogenesis in Myocardial Infarction

Stem Cells International ◽

10.1155/2018/3290372 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 40

Author(s):

Teng Ma ◽

Yueqiu Chen ◽

Yihuan Chen ◽

Qingyou Meng ◽

Jiacheng Sun ◽

...

Keyword(s):

Myocardial Infarction ◽

Endothelial Cells ◽

Real Time ◽

Real Time Pcr ◽

Target Gene ◽

Tube Formation ◽

Luciferase Reporter ◽

Reporter Assay ◽

Ischemic Diseases

Background. To cure ischemic diseases, angiogenesis needs to be improved by various strategies in ischemic area. Considering that microRNA-132 (miR-132) regulates endothelial cell behavior during angiogenesis and the safe and efficacious delivery of microRNAs in vivo is rarely achieved, an ideal vehicle for miR-132 delivery could bring the promise for ischemic diseases. As a natural carrier of biological molecules, exosomes are more and more developed as an ideal vehicle for miRNA transfer. Meanwhile, mesenchymal stem cells could release large amounts of exosomes. Thus, this study aimed to investigate whether MSC-derived exosomes can be used for miR-132 delivery in the treatment of myocardial ischemia. Methods. MSC-derived exosomes were electroporated with miR-132 mimics and inhibitors. After electroporation, miR-132 exosomes were labelled with DiI and added to HUVECs. Internalization of DiI-labelled exosomes was examined by fluorescent microscopy. Expression levels of miR-132 in exosomes and HUVECs were quantified by real-time PCR. The mRNA levels of miR-132 target gene RASA1 in HUVECs were quantified by real-time PCR. Luciferase reporter assay was performed to examine the targeting relationship between miR-132 and RASA1. The effects of miR-132 exosomes on the angiogenic ability of endothelial cells were evaluated by tube formation assay. Matrigel plug assay and myocardial infarction model were used to determine whether miR-132 exosomes can promote angiogenesis in vivo. Results. miR-132 mimics were effectively electroporated and highly detected in MSC-derived exosomes. The expression level of miR-132 was high in HUVECs preincubated with miR-132 mimic-electroporated exosomes and low in HUVECs preincubated with miR-132 inhibitor-electroporated exosomes. The expression level of RASA1, miR-132 target gene, was reversely correlated with miR-132 expression in HUVECs pretreated with exosomes. Luciferase reporter assay further confirmed that RASA1 was a direct target of miR-132. Exosomes loaded with miR-132, as a vehicle for miRNA transfer, significantly increased tube formation of endothelial cells. Moreover, subcutaneous injection of HUVECs pretreated with miR-132 exosomes in nude mice significantly increased their angiogenesis capacity in vivo. In addition, transplantation of miR-132 exosomes in the ischemic hearts of mice markedly enhanced the neovascularization in the peri-infarct zone and preserved heart functions. Conclusions. The findings suggest that the export of miR-132 via MSC-derived exosomes represents a novel strategy to enhance angiogenesis in ischemic diseases.

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Identification of upstream miRNAs of SNAI2 and their influence on the metastasis of gastrointestinal stromal tumors

Cancer Cell International ◽

10.1186/s12935-019-1006-8 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Jie Ding ◽

Yu Xia ◽

Zhaoyan Yu ◽

Jing Wen ◽

Zhuxue Zhang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Gastrointestinal Stromal Tumors ◽

Luciferase Reporter ◽

Invasion Assay ◽

Reporter Assay ◽

Mirna Microarray ◽

Stromal Tumors ◽

Dual Luciferase Reporter Assay

Abstract Background SNAI2, a member of the snail zinc finger protein family, plays an important role in the metastasis of several types of carcinoma. Objective This study aims to investigate the upstream miRNAs of SNAI2 and their influence on the metastasis of gastrointestinal stromal tumors (GISTs). Methods The expression levels of SNAI2, CDH1, and CDH2 in GISTs were determined by immunohistochemistry, and the correlations with their clinicopathologic characteristics were analyzed. Subsequently, the miRNAs involved in regulating SNAI2 expression were predicted by bioinformatics technique, screened by miRNA microarray tests, and verified by real-time PCR, dual luciferase reporter assay, and invasion assay. The influence of SNAI2 and miRNAs on the invasive ability of the GIST cells and the related mechanism were detected. Outcomes SNAI2 expression significantly increased and CDH1 expression markedly decreased in the cases of GISTs with distant metastasis. Silencing of the SNAI2 gene impaired the invasiveness of GIST cells in vitro. MiR-200b-3p, miR-30c-1-3P, and miR-363-3P were verified as the upstream metastasis-associated miRNAs of SNAI2 in GISTs by miRNA microarray, real-time PCR, dual luciferase reporter assay, and invasion assay. They bound to the 3′-UTR of SNAI2, downregulated SNAI2 expression, and inhibited the invasiveness of GIST cells. SNAI2 targetedly bound to the promoter of the CDH1 gene, downregulated the expression of CDH1, and contributed to the metastasis of GISTs. Conclusion SNAI2 and CDH1 correlated with the metastasis of GISTs, and silencing of the SNAI2 gene impaired the invasiveness of GIST cells. MiR-200b-3p, miR-30c-1-3P, and miR-363-3P contribute to the metastasis of GISTs in vitro by mediating the SNAI2/CDH1 axis. SNAI2 may be a potential target for the treatment of GISTs in the future.

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MicroRNA-513b-5p targets COL1A1 and COL1A2 associated with the formation and rupture of intracranial aneurysm

Scientific Reports ◽

10.1038/s41598-021-94116-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zheng Zheng ◽

Yan Chen ◽

Yinzhou Wang ◽

Yongkun Li ◽

Qiong Cheng

Keyword(s):

Cell Death ◽

Intracranial Aneurysm ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Control Group ◽

Type I ◽

Mrna Levels ◽

Reporter Assay ◽

Over Expression ◽

Tnf Α

AbstractCollagen-type I alpha 1 chain (COL1A1) and COL1A2 are abnormally expressed in intracranial aneurysm (IA), but their mechanism of action remains unclear. This study was performed to investigate the mechanism of COL1A1 and COL1A2 affecting the occurrence and rupture of IA. Quantitative real-time polymerase chain reaction was used to measure the expression of hsa-miR-513b-5p, COL1A1, COL1A2, TNF-α, IL-6, MMP2, MMP3, MMP9 and TIMP4 in patients with ruptured IA (RA) (n = 100), patients with un-ruptured IA (UA) (n = 100), and controls (n = 100). Then, human vascular smooth muscle cells (HASMCs) were cultured, and dual luciferase reporter assay was performed to analyse the targeting relationship between miR-513b-5p and COL1A1 or COL1A2. The effects of the miR-513b-5p mimic and inhibitor on the proliferation, apoptosis, and death of HASMC and the RIP1-RIP3-MLKL and matrix metalloproteinase pathways were also explored. The effect of silencing and over-expression of COL1A1 and COL1A2 on the role of miR-513b-5p were also evaluated. Finally, the effects of TNF-α on miR-513b-5p targeting COL1A1 and COL1A2 were tested. Compared with those in the control group, the serum mRNA levels of miR-513b-5p, IL-6 and TIMP4 were significantly decreased in the RA and UA groups, but COL1A1, COL1A2, TNF-α, IL-1β, MMP2, MMP3 and MMP9 were significantly increased (p < 0.05). Compared with those in the UA group, the expression of COL1A1, COL1A2, TNF-α, IL-1β and MMP9 was significantly up-regulated in the RA group (p < 0.05). Results from the luciferase reporter assay showed that COL1A1 and COL1A were the direct targets of miR-513b-5p. Further studies demonstrated that miR-513b-5p targeted COL1A1/2 to regulate the RIP1-RIP3-MLKL and MMP pathways, thereby enhancing cell death and apoptosis. Over-expression of COL1A1 or COL1A2, rather than silencing COL1A1/2, could improve the inhibitory effect of miR-513b-5p on cell activity by regulating the RIP1-RIP3-MLKL and MMP pathways. Furthermore, over-expression of miR-513b-5p and/or silencing COL1A1/2 inhibited the TNF-α-induced cell proliferation and enhanced the TNF-α-induced cell death and apoptosis. The mechanism may be related to the inhibition of collagen I and TIMP4 expression and promotion of the expression of RIP1, p-RIP1, p-RIP3, p-MLKL, MMP2 and MMP9. MiR-513b-5p targeted the inhibition of COL1A1/2 expression and affected HASMC viability and extracellular mechanism remodelling by regulating the RIP1-RIP3-MLKL and MMP pathways. This process might be involved in the formation and rupture of IA.

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Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo

PeerJ ◽

10.7717/peerj.10374 ◽

2020 ◽

Vol 8 ◽

pp. e10374

Author(s):

Ying Jin ◽

Xiaoyan Sun ◽

Fang Pei ◽

Zhihe Zhao ◽

Jeremy Mao

Keyword(s):

Bone Formation ◽

Mrna Expression ◽

Nuclear Translocation ◽

Luciferase Activity ◽

Fracture Repair ◽

Luciferase Reporter ◽

Control Group ◽

Type I ◽

Alizarin Red ◽

Osteogenic Induction

Background Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. Methods We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. Results Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. Conclusion Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

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The Differential Expression of miRNAs and a Preliminary Study on the Mechanism of miR-194-3p in Keloids

BioMed Research International ◽

10.1155/2019/8214923 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Zhishan Xu ◽

Bingyu Guo ◽

Peng Chang ◽

Qiang Hui ◽

Wei Li ◽

...

Keyword(s):

Western Blot ◽

Real Time ◽

Differential Expression ◽

Real Time Pcr ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Cultured Fibroblasts ◽

Gene Assay ◽

And Migration ◽

Related Proteins

The aim of this study was to detect abnormally expressed microRNA (miRNA) in keloids and to study their functions. The differential expression of miRNAs in keloids and normal tissue was detected by gene microarray. MiRNA expression was verified by real-time PCR. A luciferase reporter gene assay, western blot, and real-time PCR were used to detect the effect of miR-194-3p on RUNX2. An MTT assay and a transwell assay were used to detect the effect of miR-194-3p in both primary cultured fibroblasts and HKF cells. Related proteins were analysed by western blot and real-time PCR. The expression of miR-194-3p was lower in keloids, and MiR-194-3p was shown to target RUNX2 directly. MiR-194-3p inhibited the proliferation and migration of fibroblasts through the inhibition of CDK4 and MMP2. MiR-194-3p and RUNX2 may become new targets for the prevention and treatment of keloids.

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WT1 and PRAME Expression in CML: Quantitation by Real-Time PCR and Correlation with BCR-ABL Expression Levels

Blood ◽

10.1182/blood.v112.11.4225.4225 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4225-4225

Author(s):

Gareth Gerrard ◽

Caroline Cloke ◽

Ian H Gabriel ◽

Katayoun Rezvani ◽

Jane F. Apperley ◽

...

Keyword(s):

Tyrosine Kinase ◽

Real Time ◽

Real Time Pcr ◽

Residual Disease ◽

Philadelphia Chromosome ◽

Treatment Modalities ◽

Horizontal Line ◽

Over Expression ◽

Diagnostic Samples

Abstract Introduction. Chronic myeloid leukaemia (CML) is a malignant clonal myeloproliferative disorder characterised by the presence of the Philadelphia chromosome (Ph) and expression of the BCR-ABL fusion protein with constitutive tyrosine kinase activity and multi-target dysregulation. Standard therapy employs the use of tyrosine kinase inhibitors, such as Imatinib mesylate, with a high level of success. However, in a proportion of patients this approach is sub-optimal necessitating the use other treatment modalities. Allogeneic stem cell transplantation (allo-SCT) is established and potentially curative via the immunologically mediated graft-versus-leukaemia (GvL) effect, but it is associated with significant morbidity, long term effects and high mortality rate (30%). An alternative approach is to employ peptide vaccines directed against leukaemia associated antigens (LAA) in order to evoke a curative anti-leukaemia immune response without the risk of graft versus host disease. Prevalent among these LAAs are WT1 (Wilms Tumour 1) and PRAME (preferentially expressed antigen in melanoma). WT1 encodes a transcription factor of the zinc-finger family, and is known to be over-expressed in many types of leukaemia. PRAME (PRA) is a cancer testis antigens over-expressed in haematological malignancies including CML, making it an attractive target for immunotherapy. HLA-A2 and A-24 restricted epitopes derived from PRA have been shown to be immunogenic by a number of groups. Here, we studied the value of PRA and WT1 measurement in assessing minimal residual disease (MRD) in CML compared to BCR-ABL. Materials & Methods. cDNA from PB samples from 76 diagnostic samples and 127 follow-up samples from 114 patients were tested. The follow-up samples were subdivided into 3 groups according to BCR-ABL expression: MRD1 >10%, MRD2: 1%-10%, MRD3 <1%. All samples were analysed for WT1 and PRA, except for the diagnostic samples, where only 6 were available for PRA analysis due to a lack of material. All expression data was obtained by quantitative real-time PCR (Q-PCR) on the ABI 7500 platform. The BCR-ABL was expressed as a percentage ratio against ABL, using absolute quantification. WT1 and PRA expression was derived using the relative expression dCt method, normalised against a house keeping gene (GUS), after ensuring equivalent Q-PCR efficiencies. Results. WT1 and PRA expression was detectable in all samples analysed. A non-parametric correlation, performed on all data points, found a positive correlation between expression of BCR-ABL and expression of WT1 (r= 0.6710, P<0.0001) and PRA (r= 0.4194, P<0.0001) (Figure 1). The median WT1 and PRA expression of the remission samples was calculated and used as a cut-off to define over-expression (2.89E-05 and 7.04E-05, respectively). This was then used to assess the proportion of samples from the diagnostic and follow-up groups that over-expressed WT1 (Diag= 96.1%, MRD1= 90.0%, MRD2= 66.7%, MRD3= 33.3%) and PRA (Diag= 100%, MRD1= 82.5%, MRD2= 41.7%, MRD3= 60.0%). The differences in over-expression between the groups were found to be highly significant by 2×4 contingency Chi square test (P<0.0001 for both WT1 and PRA). Discussion. This analysis shows that both WT1 and PRA expression positively correlate with BCR-ABL level in CML patients, and can be reliably quantitated using Q-PCR. This technique can therefore provide useful information for treatment monitoring of immunotherapy directed against such antigens. The median expression of the remission samples can be used as a useful cut-off for assessing over-expression for WT1, but maybe of less value in respect to PRA, due to a lack of discrimination at low BCR-ABL levels shown by the discordantly high value of positivity in the MRD3 group. Figure 1. Expression of A: WT1 and B: PRAME in CML, subdivided by BCR-AEL expression (vertical lines) and over-expression thresold (horizontal line). Figure 1. Expression of A: WT1 and B: PRAME in CML, subdivided by BCR-AEL expression (vertical lines) and over-expression thresold (horizontal line).

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Downregulation of Prdx2 Protects Osteoporosis Rats by Regulating Receptor Activator of Nuclear Factor Kappa-B/Osteoprotegerin Pathway

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2019.2042 ◽

2019 ◽

Vol 9 (6) ◽

pp. 839-844

Author(s):

Shuai Zhang ◽

Weiwei Guo ◽

Xin Zhao ◽

Peng Li

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Structural Changes ◽

Biomechanical Properties ◽

Nodule Formation ◽

Sham Operation ◽

Mineral Density ◽

Alizarin Red ◽

Sham Operation Group ◽

Metabolic Characteristics

Osteoporosis (OP) is a bone disease caused by various causes and can be found in various stages, such as juvenile, adult, menopausal and old age. OP has the systemic, degenerative, and metabolic characteristics, which leads to loss of bone mass, structural changes, and biomechanical properties degeneration. Prdx2 is a member of the peroxiredoxase family with antioxidant effects and involved in the regulation of bone and joint diseases. However, the role of Prdx2 in OP and related mechanisms has not been elucidated. SD rats were randomly divided into 2 groups, OP group in which OP rat model was prepared by ovariectomy and sham operation group. Prdx2 siRNA was transfected into OP rats followed by analysis of the expression of Prdx2, Opn, and Osterix by real-time PCR, bone density changes by dual energy line bone densitometer, formation of mineralized nodules by Alizarin red staining, Serum osteocalcin (OC) activity by ELISA as well as RANK and osteoprotegerin (OPG) expressions by real-time PCR and Western blot. Prdx2 expression was significantly increased, bone mineral density (BMD) was reduced, mineralized nodule formation was attenuated, Opn and Osterix levels were downregulated, together with decreased serum OC activity, increased RANK expression, and declined OPG expression apparently in OP rats compared with sham group (P < 0.05). Prdx2 siRNA transfection downregulated Prdx2 and RANK levels, increased BMD, mineralized nodule formation, Opn, OPG, and Osterix levels, as well as serum OC activity in OP rats (P < 0.05). Prdx2 expression is elevated in osteoporotic rats, which is associated with decreased osteogenic differentiation and BMD, leading to osteoporosis. Downregulation of Prdx2 expression can improve osteoporosis by regulating the RANK/OPG pathway.

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LncRNA ACTA2-AS1 Suppress Colon Adenocarcinoma Progression by Sponging miR-4428 Upregulation BCL2L11

10.21203/rs.3.rs-113478/v1 ◽

2020 ◽

Author(s):

Qingyun Pan ◽

Ying Huang ◽

Yirui Wang ◽

Deke Li ◽

Changjiang Lei

Keyword(s):

Cell Proliferation ◽

Colony Formation ◽

Colon Adenocarcinoma ◽

Human Colon ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Over Expression ◽

Non Coding Rna

Abstract BackgroundLong non-coding RNA is considered to be essential to modulate the development and progression of human malignant cancers. And long non-coding RNA can act as crucial modulators by sponging the corresponding microRNA in tumorigenesis. We aimed to elucidate the function of ACTA2-AS1 and its molecular mechanism in colon adenocarcinoma. Materials and MethodsThe expression of ACTA2-AS1, miR-4428 and BCL2L11 in colon adenocarcinoma tissues were detected via qRT-PCR. SW480 and HT29 cells were transfected with shRNA ACTA2-AS1, OE ACTA2-AS1, miRNA mimics of miR-4428, miR-4428 inhibitor, si-BCL2L11 and over-expression of si-BCL2L11. Cell proliferation, colony formation and apoptosis were respectively assessed using CCK-8 assay, colony assay and flow cytometry. Luciferase reporter assay was performed to verify the targets of ACTA2-AS1 and miR-4428. Tumor subcutaneous xenograft mode was constructed to explore tumor growth in vivo. ResultsACTA2-AS1 was obviously downregulated in human colon adenocarcinoma tissues and colon adenocarcinoma cell lines. Silence or over-expression of ACTA2-AS1 promoted or inhibited cell proliferation and colony formation abilities, and regulated apoptosis. The silence of ACTA2-AS1 resulted in the decrease of Bax and increase of Bal2, while restored in OE ACTA2-AS1 group when compared with the control transfected cells. In addition, luciferase reporter assay revealed that ACTA2-AS1 interacted with miR-4428 and suppressed its expression. miR-4428 could bind to 3ʹ untranslated region of BCL2L11 and modulated the expression of BCL2L11 negatively. Knockdown of ACTA2-AS1 and over-expression of BCL2L11 reversed the biological function that ACTA2-AS1 mediated by knockdown ACTA2-AS1 alone. ConclusionOur data demonstrated that ACTA2-AS1 could suppress colon adenocarcinoma progression via sponging miR-4428 to regulate BCL2L11 expression.

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Osteogenic Differentiation of Pre-Conditioned Bone Marrow Mesenchymal Stem Cells with Nisin on the Modified Poly-L-Lactic-Acid Nanofibers

10.21203/rs.3.rs-553590/v1 ◽

2021 ◽

Author(s):

Fariba Sadraei ◽

Marzieh Ghollasi ◽

Fatemeh Khakpai ◽

Raheleh Halabian

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Lactic Acid ◽

Osteogenic Differentiation ◽

Real Time ◽

Real Time Pcr ◽

Alizarin Red ◽

Marrow Mesenchymal Stem Cells

Abstract Background: Human bone marrow-derived mesenchymal stem (MSCs) cells are undifferentiated cells with the self-renewing ability and multi-lineage differentiation beneficial for regenerative medicine. Nano scaffolds are novel materials employed in bone repair and regeneration. Nisin is a prebiotic that can increase stem cells’ life span and proliferation. This study attempted to provide a proper strategy for bone marrow mesenchymal stem cells differentiation into the Osteocytes on a Poly‐L‐lactic‐acid scaffold (PLLA) after pretreating with probiotic Nisin. Methods: MSC osteogenic differentiation was evaluated by measuring Calcium, Alkaline phosphatase, and quantitative tests such as Real-Time PCR, Acridine Orange, Alizarin Red, Von Kossa, and others. Results: The result of the MTT test showed that the optimal dose of Nisin probiotic for the MSCs’ preconditioning was 200 IU/mL on the 1st, 3rd, and 5th days of culture. Real-time PCR data indicated that the expression rate of ALP, Osteonectin, Osteocalcin, and Collagen I have increased in the presence of Nisin, while the RUNX-2 gene expression has decreased. Furthermore, the results of Alizarin Red and Von Kossa tests, as well as Scanning electron microscopy (SEM), revealed that the cell proliferation in the preconditioned samples with Nisin increased significantly. Conclusions: The study concluded that the cell proliferation and differentiation increased in samples pretreated with Nisin on the PLLA Nano scaffolds.

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MicroRNA Hsa-Let-7b Regulates the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting CTHRC1

Stem Cells International ◽

10.1155/2021/5791181 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Lin Fu ◽

Na Li ◽

Yu Ye ◽

Xiaying Ye ◽

Tong Xiao ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Periodontal Ligament ◽

Cell Counting ◽

Luciferase Reporter ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Proliferation Ability ◽

Polymerase Chain ◽

Dual Luciferase Reporter Assay

Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells’ (MSCs’) biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1.

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